Research conducted over the past 50 years has provided considerable knowledge regarding the chemical constituents of smokeless tobacco products, smoked tobacco products and cigarette smoke. Meanwhile, there is a paucity of data regarding the microbial constituents of tobacco and smoke, and their associated adverse health effects. Yet, tobacco microbial constituents may play significant roles in the development of both infectious and chronic diseases among tobacco users. To address these knowledge gaps, the longterm objective of this proposed project is to harness the power of next-generation sequencing technologies to increase our understanding of the microbial constituents of conventional, new and emerging tobacco products, and greatly improve our understanding of microbial-related health risks among tobacco users. The specific aims of the proposal are as follows: 1) To explore the bacterial microbiome of conventional, new and manipulated smoked and smokeless tobacco products and smoke, and examine the role of specific genera in tobacco-specific N-nitrosamine formation; 2) To provide novel, baseline data on the composition of the oral microbiome and its associated expressed activities in smokers and smokeless tobacco users compared with that of non-users; and 3) To characterize the transient changes-bacterial community composition and expressed metabolic activities-in the oral microbiome after single-use of new and manipulated smoked and smokeless tobacco products. To accomplish these aims, this study will employ an innovative, polyphasic approach involving the application of next-generation sequencing methods in: 1) time course experiments with conventional, new and manipulated tobacco products; 2) a longitudinal study of tobacco users and nonusers; and 3) cross-over trials of tobacco users testing new or manipulated tobacco products. This approach will generate a rich, novel and valuable data source that can be immediately utilized by FDA as it implements the Family Smoking Prevention and Tobacco Control Act and seeks to improve tobacco regulation for the benefit of public health. Specifically, these data could inform potential new microbial-related tobacco regulations that have never before been considered by U.S. governmental regulators.